Enterovirulent <Emphasis Type="Italic">E. coli</Emphasis> in inflammatory and noninflammatory bowel diseases

We determined the incidence of enterovirulent E. coli (EVEC; which can to cause gastrointestinal infections) in strains isolated from patients with both of the major inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) and from patients with noninflammatory bowel diseases (nonIBD). Cell detachment E. coli (CDEC) were detected in 14 % of all strains. A significant difference in the presence of CDEC was found between the groups of strains isolated from UC (24.1 %), nonIBD (11.9 %) and CD (4.7 %). Enteroaggregative E. coli (EAggEC) were detected in 2.5 %, typical enteropathogenic strains (EPEC) in 1.3 % and enterotoxigenic ones (ETEC) in 1.5 %. Enteroinvasive (EIEC) and shigatoxin producing E. coli (STEC) were not detected. Some strains showed a high invasion level in gentamicin-protection assay. These strains could therefore belong to adherent-invasive E. coli (AIEC) because they are free of genes encoding invasins (ipaH, ial) and are equipped with fimA gene. However, complete characterization of these strains and their classification as AIEC will require further tests.     

Interferon‐gamma production in Lyme arthritis synovial tissue promotes differentiation of fibroblast‐like synoviocytes into immune effector cells

Lyme arthritis (LA), a late disease manifestation of Borrelia burgdorferi infection, usually resolves with antibiotic therapy. However, some patients develop proliferative synovitis lasting months to several years after spirochetal killing, called postinfectious LA. In this study, we phenotyped haematopoietic and stromal cell populations in the synovial lesion ex vivo and used these findings to generate an in vitro model of LA using patient‐derived fibroblast‐like synoviocytes (FLS). Ex vivo analysis of synovial tissue revealed high abundance of IFNγ‐producing T cells and NK cells. Similar to marked IFNγ responses in tissue, postinfectious LA synovial fluid also had high levels of IFNγ. HLA‐DR‐positive FLS were present throughout the synovial lesion, particularly in areas of inflammation. FLS stimulated in vitro with B. burgdorferi, which were similar to conditions during infection, expressed 68 genes associated primarily with innate immune activation and neutrophil recruitment. In contrast, FLS stimulated with IFNγ, which were similar to conditions in the postinfectious phase, expressed >2,000 genes associated with pathogen sensing, inflammation, and MHC Class II antigen presentation, similar to the expression profile in postinfectious synovial tissue. Furthermore, costimulation of FLS with B. burgdorferi and IFNγ induced greater expression of IL‐6 and other innate immune response proteins and genes than with IFNγ stimulation alone. These results suggest that B. burgdorferi infection, in combination with IFNγ, initiates the differentiation of FLS into a highly inflammatory phenotype. We hypothesise that overexpression of IFNγ by lymphocytes within synovia perpetuates these responses in the postinfectious period, causing proliferative synovitis and stalling appropriate repair of damaged tissue.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

ESX‐1 exploits type I IFN‐signalling to promote a regulatory macrophage phenotype refractory to IFNγ‐mediated autophagy and growth restriction of intracellular mycobacteria

The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ‐signalling in macrophages. Still, the host–pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX‐1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN‐signalling to promote an IL‐12^low/IL‐10^high regulatory macrophage phenotype characterized by secretion of IL‐10, IL‐27 and IL‐6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ‐mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ‐refractory phenotype was partly mediated by IL‐27‐signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage‐modulating function for the ESX‐1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.     

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

Background  

Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.     

Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn''s disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH _LF82 (expressing FimH with an AIEC-associated mutation) with fimH _K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.     

Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT‐29 cells and also down‐regulates enteropathogen‐induced tumor necrosis factor‐α and interleukin‐8 expression

The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT‐29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT‐29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT‐29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor‐α and interleukin‐8 expression was analyzed in HT‐29 cells stimulated with E. coli using qRT‐PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT‐29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor‐α and interleukin‐8 expression in HT‐29 cells stimulated with E. coli.     

Adherent-invasive Escherichia coli, strain LF82 disrupts apical junctional complexes in polarized epithelia

Background  

Although bacteria are implicated in the pathogenesis of chronic inflammatory bowel diseases (IBD), mechanisms of intestinal injury and immune activation remain unclear. Identification of adherent-invasive Escherichia coli (AIEC) strains in IBD patients offers an opportunity to characterize the pathogenesis of microbial-induced intestinal inflammation in IBD. Previous studies have focused on the invasive phenotype of AIEC and the ability to replicate and survive in phagocytes. However, the precise mechanisms by which these newly identified microbes penetrate the epithelial lining remain to be clarified. Therefore, the aim of this study was to delineate the effects of AIEC, strain LF82 (serotype O83:H1) on model polarized epithelial monolayers as a contributor to intestinal injury in IBD.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

A role for vimentin in Crohn disease

Crohn disease (CD), one of the major chronic inflammatory bowel diseases, occurs anywhere in the gastrointestinal tract with discontinuous transmural inflammation. A number of studies have now demonstrated that genetic predisposition, environmental influences and a dysregulated immune response to the intestinal microflora are involved. Major CD susceptibility pathways uncovered through genome-wide association studies strongly implicate the innate immune response (NOD2), in addition to the more specific acquired T cell response (IL23R, ICOSLG) and autophagy (ATG16L1, IRGM). Examination of the disease-associated microbiome, although complex, has identified several potentially contributory microorganisms, most notably adherent-invasive E.coli strains (AIEC), which have been isolated by independent investigators in both adult and pediatric CD patients. Here we discuss our recent finding that the type-III intermediate filament (IF) protein VIM/vimentin is a novel NOD2 interacting protein that regulates NOD2 activities including inflammatory NFKB1 signaling, autophagy and bacterial handling.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome‐dependent secretion of IL‐1β and the danger signals ASC and HMGB1

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger‐associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro‐inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF‐κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase‐1 and stimulates secretion of mature IL‐1β. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high‐mobility group box 1 protein and apoptosis‐associated speck‐like protein, with a similar time‐course as caspase‐1 activation. Thus, F. nucleatum expresses the pathogen‐associated molecular patterns necessary to activate NF‐κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.     

Understanding the cause of false negative β‐d‐glucuronidase reactions in culture media containing fermentable carbohydrate

Aims:  To explain the basis for false negative β‐glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Methods and Results:  Escherichia coli strains were assessed for their reactions in culture media containing a β‐d ‐glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β‐d ‐glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β‐d ‐glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose‐containing media expressed virtually no β‐d ‐glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. Conclusions:  E. coli that failed to produce green colonies on MLGA produced lower levels of β‐d ‐glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Significance and Impact of the Study:  Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β‐d ‐glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).